The present invention relates generally to protein chemistry of analogs of granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin-3 (IL-3) proteins, fusion proteins comprising GM-CSF and IL-3, and more particularly to a process for purifying such analogs and fusion proteins.
The differentiation and proliferation of hematopoietic cells is regulated by secreted glycoproteins collectively known as colony-stimulating factors (CSFs). In humans, these proteins include granulocyte-macrophage CSF (GM-CSF), which promotes granulocyte and macrophage production from normal bone marrow, and which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. Interleukin-3 (IL-3; also known as multi-CSF) also stimulates formation of a broad range of hematopoietic cells, including granulocytes, macrophages, eosinophils, mast cells, megakaryocytes and erythroid cells. GM-CSF and IL-3 thus have considerable overlap in their broad range of biological activities.
The biological activities of GM-CSF and IL-3 are mediated by binding to specific cell surface receptors expressed on prime cells and in vitro cell lines. Certain of these cell surface receptors bind GM-CSF alone, while others bind IL-3 alone. There also appears to be a class of receptor that binds both IL-3 and GM-CSF (Park et al., J. Biochem. 264:5420, 1989), referred to as GM-CSF/IL-3 receptor.
Fusion proteins comprising GM-CSF and IL-3 and DNA sequences encoding such fusion proteins are described in U.S. Pat. Nos. 5,073,627 and 5,108,910, respectively, both of which are incorporated by reference herein. The fusion proteins are more biologically active than GM-CSF or IL-3 alone or in combination and, relative to IL-3, have a significantly higher affinity of binding to cell lines that express GM-CSF/IL-3 receptors as compared to cell lines with receptors that bind only IL-3 or GM-CSF.
U.S. Pat. Nos. 5,073,627 and 5,108,910 also disclose methods of purifying the fusion proteins. The methods disclosed involve a combination of ion exchange chromatography steps and reverse-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC is difficult to scale-up to a level achieving commercially significant yields of proteins, and additionally requires use of organic solvents. The organic solvents present a number of safety hazards, from the generation of noxious and harmful fumes to risk of explosion. Furthermore, organic solvents require implementation of hazardous waste disposal procedures, and are more likely to result in denaturation or inactivation of biological material than procedures which more nearly mimic physiologic conditions.
Ion exchange chromatography, usually entailing a series of ion exchange steps, may be used to purify proteins. The number and sequence of ion exchange steps, and the optimal purification conditions, are usually determined empirically for individual proteins. The steps and conditions necessary for purification of a fusion protein cannot be predicted by analyzing the physicochemical properties of the proteins from which the fusion protein is derived. As an example of this, the recombinant fusion proteins described herein, which comprise GM-CSF and IL-3, can be adsorbed to an anion exchange resin such as a resin having pendant diethylaminoethyl (DEAE) groups, and eluted at a fairly acidic pH, whereas IL-3 would not be adsorbed to an anion exchange resin having pendant DEAE groups under these conditions.
Therefore, there is a need in the art to develop methods of purifying fusion proteins comprising GM-CSF and IL-3 that circumvent the use of RP-HPLC and are amenable to scaleup to the level required for commercial success.